1. Field of the Invention
The present invention relates to methods for determining the impact of diluting a solution. In particular, the present invention relates to a method for determining a volume of a solution mixed with a diluent. The present invention also relates to a method for determining the efficiency of removing one or more reagents from a vessel using a wash solution. The present invention further relates to a method for determining the dilution ratio for a solution under dilution.
2. Description of the Prior Art
Existing versions of the Multichannel Verification System (“MVS®”), which are commercially available from Artel, Inc. (“Artel”) of Westbrook, Me. and are the subject of U.S. Pat. No. 6,741,365 and U.S. Pat. No. 7,187,455, include the use of equipment and aqueous sample solutions to determine the accuracy of dispensing devices over specified volume ranges. Additionally, an existing method which may be used to measure the volume of a non-aqueous sample solution created from an aqueous precursor is the subject of US Patent Application Publication No. 2007/0141709. The entire contents of U.S. Pat. No. 6,741,365, U.S. Pat. No. 7,187,455 and US Patent Application Publication No. 2007/0141709 are incorporated herein by reference. Publication No 2007/0141709 describes a general method for creating test solutions from aqueous MVS® stock solutions and core calculation procedures correspond to those described in U.S. Pat. Nos. 6,741,365 and 7,187,455. The aqueous-based solutions offered in the existing versions of the MVS® system meet the needs of many users, and the method for creating test solutions from non-aqueous solvents described in Publication No. 2007/0141709 also offers a test method needed by many users.
However, there are limitations to the MVS® system and to the method of Publication No. 2007/0141709. For example, there are limitations to the types of solvents that may be used to create test solutions from MVS® stock solutions while still maintaining accurate calculations following the mathematical approaches described in U.S. Pat. Nos. 6,741,365 and 7,187,455. Namely, any solvent that significantly alters the solution meniscus in the microtiter well can have a detrimental effect on the results calculated by said approaches. The MVS® methods described in U.S. Pat. Nos. 6,741,365 and 7,187,455 are based on a generally flat meniscus, a controlled or known solution chemistry that yields a reproducible meniscus, and/or a correction factor which accounts for minor deviations in meniscus shape.
When an uncharacterized solvent included in a new test solution from MVS® stock solutions (as described in Publication No. 2007/0141709) and the existing MVS® methods (as described in U.S. Pat. Nos. 6,741,365 and 7,187,455) are applied, there can be substantial errors, depending on how curved the meniscus becomes. The existing MVS® methods rely upon knowledge of this meniscus curvature in order to accurately return results on the volume delivered to the microtiter well, and can only do so through application of a correction factor. Thus, for solvents that have not been characterized for their affect on the solution meniscus, the results produced by the existing MVS® methods are less accurate than they could be. A method to cancel out the impact of variations in meniscus would be useful in determining the volume of a solution dispensed.
Various types of assays performed in life science and pharmaceutical laboratories require wash steps to remove unwanted or used reagents from a reaction vessel. For example, Enzyme-linked Immunosorbent Assays (“ELISA assays”) require introduction of a tagged ligand. This tagged ligand binds to the molecular entity of interest, if it is present, and can be measured. The measurement is often a fluorometric, photometric or radiometric measurement, depending on the type of tag used. However, before the measurement step can occur, all unbound tagged-ligand must be removed from the reaction vessel by a rinse, or wash, step with a wash solution, often consisting of buffered water.
For assays conducted in microtiter plates, wash steps are commonly employed to exchange the solution within the wells. These wash steps are carried out using specialized equipment, called plate washers. Some examples of plate washers include the Tecan Power Washer 384 (Tecan US, Durham, N.C.) and the Biotek ELx405 Microplate Washer (BioTek Instruments, Winooski, Vt.).
Plate washers typically operate by dispensing a wash solution into the wells of the microtiter plate, while at the same time removing solution. Thus, they incorporate a dispense tube, and an aspirate tube, often side-by-side. The solution is thus flowed into and out of the well at a high velocity in order to effectively flush out unwanted reagents. The wash solution is dispensed into the wells by the dispense tip, which is connected by tubing to a large volume reservoir (often greater than 1 L) containing clean wash solution. The aspirate tip is connected via tubing to a waste reservoir. The dispense tip is often inserted into the well near the bottom, thus introducing wash solution at the bottom of the well. Conversely, the aspirate tip is inserted at a height near the top ⅓ of the well height. Positioning the aspirate tip at this height forces the well contents to be pushed by the wash solution, flowing into the well near the bottom, up towards the top of the well where it is removed by the aspiration tip. When the wash procedure is complete, the total solution height is equal to the height of the aspirate tip with respect to (or above) the plate bottom. This height thus determines the total volume of solution that will be left behind in the well.
For many microtiter plate-based assays incorporating a wash step, a quantitative understanding of the efficiency of washing is not needed. However, under some circumstances an understanding of how efficiently reagents are being removed from the wells by the plate washer is needed. One way to measure washing efficiency is to determine the degree of dilution that has occurred for the reagents in the wells. For such testing, the dilution testing scheme taught by existing methods can be used. However these existing methods require the use of a diluent solution to be added to the wash reservoir. In many cases, adding such a large volume of diluent is inconvenient and costly, especially considering a dead volume needed to fill the lines of 200-500 mL. Thus, an improved method for testing plate washing efficiency is desired.
Many test procedures carried out in life science and pharmaceutical laboratories also require dilution-based volume transfer steps, such as dose response and detection limit assays, for example. For many of these procedures, quantitative measurements are collected and decisions are made based upon an assumed, rather than a measured, dilution ratio. Often these assumptions are based upon a potentially misplaced trust in the performance of automated liquid delivery equipment. Accurately knowing sample concentration is critical for properly interpreting the experimental results, which can only be obtained if the experimental dilution ratio is known and controlled. Thus, the ability to accurately measure each dilution step in a dilution procedure having a plurality of dilution steps is required for proper assay analysis.
What is needed is a method that enables accurate calculation of the volume of a solution dispensed independent of meniscus. What is also needed is an effective method to determine the efficiency of plate washing routines. Further, what is needed is a method to determine dilution accuracy in a dilution procedure including a plurality of dilution steps.